Personalizing substance for application to the skin or addition to tattoo ink and methods of preparation thereof

ABSTRACT

Compositions for delivering materials, such as a biological material, sand, soil, metal, water, sea water, holy water, synthetic or biological polymers, cremated ash, ceramics, animal or plant tissue, or another physiologically compatible component having personal significance to an individual are described herein. The material(s) are encapsulated in an inert, non-bioerodible, hydrophobic, polymeric material. Methods of making microparticles encapsulating the personalizing substance and methods of use are also provided. The personalizing substance may be encapsulated in a polymeric non-bioerodible microparticle. The encapsulated personalizing substance may be combined with a carrier for delivery to an individual&#39;s skin. In some embodiments, the personalizing substance is added to a tattoo ink and incorporated in a tattoo created on an individual&#39;s skin. Following injection in the skin, the encapsulated material remains in the microparticles, and is not released over time.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.14/593,409, filed Jan. 9, 2015, which claims priority to U.S.Provisional Patent Application No. 61/925,827 filed on Jan. 10, 2014,the contents of each of which are incorporated herein in their entirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is 121608_00010_Sequence_Listing. The size of thetext file is 1 KB, and the text file was created on Feb. 4, 2016.

FIELD OF THE INVENTION

The present invention relates to personalizing materials to be added tothe skin, such as additives for tattoo ink.

BACKGROUND OF THE INVENTION

Humans have been applying tattoos to the skin for thousands of years.For example, the first recorded formula for mixing and applying tattooink dates back to the fifth century and is attributed to the Romanphysician Aetius. Tattoo inks were derived from natural substances andcomprised a suspension of pigmented particles in a liquid carrier.Applying tattoo ink with needles or similar instruments to the skin,where the ink remains permanently, produces tattoos. This techniqueintroduces the pigment suspension through the skin by an alternatingpressure-suction action caused by the elasticity of the skin incombination with the movement of the needle. Water and other carriersfor the pigment introduced into the skin diffuse through the tissues andare absorbed. Once the skin has healed, most pigment particles remain inthe interstitial space of the tissue. During the healing process, someparticles of pigment are eliminated from the skin surface. Afterhealing, the tattoo is made up of the remaining particles of pigmentlocated in the dermis where they are engulfed by phagocytic skin cellsor are retained in the extracellular matrix. See US Publishedapplication no. 2009/0311295 to Mathiowitz et al. Inks used fortattooing resist elimination due to their inertness and the relativelylarge size of the insoluble pigment particles. A tattoo produced in thismanner will partially fade over time but will generally remain visible.Tattoos are used for a variety of reasons, primarily for ornamentationof the skin. See U.S. Pat. No. 6,013,122 to Klitzman & Koger.

Despite advances in methods of applying tattoo ink to the skin, such asthe electric tattoo ink gun, tattoo inks in commercial use today aresimilar to those used centuries ago. Standard tattoo inks contain apigment comprising metal salts dissolved in a carrier, usually ethanolor water, to disperse the pigment in the dermis. See U.S. Pat. No.6,013,122. Thus a need exists for novel formulations of tattoo ink withimproved properties.

SUMMARY OF THE INVENTION

Compositions for delivering materials, such as a biological material,sand, soil, metal, water, sea water, holy water, synthetic or biologicalpolymers, cremated ash, ceramics, animal or plant tissue, or anotherphysiologically compatible component having personal significance to anindividual are described herein. These materials may be encapsulated andthen administered to the skin of an individual to create a personalizedtattoo.

The material(s) are encapsulated in a nonbioerodible, polymericmicroparticle, wherein the microparticle comprises a biocompatible,non-bioerodible, hydrophobic polymer. Preferably the polymers(copolymers, or blends thereof) that form the microparticles have aglass transition temperature that is higher than 60° C. or have amelting point that is greater than 50° C. Methods of making and usingthe compositions are also provided.

A personalized ink tattoo creates a physical connection with a person,object, place, or event, because the personalized tattoo incorporatesinto the tattoo ink and, therefore, the image displayed in the skin, apersonalizing substance. The compositions described herein may bedelivered to a person's skin to create a personalized ink tattoo. Forexample, the compositions may be additives to a tattoo ink.Alternatively, the compositions can be delivered to the individual'sskin without adding a tattoo ink, for example, by administering thecomposition to a site where a tattoo is already present.

The composition is delivered to the individual in a suitable carrier,optionally in combination with a tattoo ink. For example, at the time ofuse, the composition may be mixed with the tattoo ink and the mixturecan be applied using a standard tattoo needle and procedure.

Alternatively, the composition is administered with a suitable carrierto one or more desired sites in an individual's skin. Typically, amarker is added to the site, or the site contains a marker, to indicatethe location of the encapsulated material.

Following delivery to an individual's skin, the encapsulated materialremains in the microparticles and the microparticles do not erode. Theencapsulated material is not released from the microparticles. Theencapsulated material remains in the microparticles for as long as it isin the individual's body, such as for at least 5 years, at least 10years, at least 15 years, at least 20 years, or for a longer period oftime.

In a preferred embodiment, the composition includes DNA obtained fromone or more humans, non-human animals, or plants of significance to theindividual, or any combination thereof. The DNA may be obtained by anystandard method, such as a non-invasive cheek-swab. The DNA isencapsulated in a non-bioerodible, polymeric microparticle, wherein themicroparticle comprises a biocompatible, hydrophobic, non-bioerodiblepolymer.

In some aspects, the invention relates to a composition comprising apersonalizing substance encapsulated in a non-bioerodible, polymericmicroparticle, wherein the microparticle comprises a biocompatible,hydrophobic, non-bioerodible polymer, and wherein the microparticle doesnot release the personalizing substance. In certain embodiments, thecomposition further comprises a carrier suitable for injection into theskin. In certain embodiments, the personalizing substance is in the formof nanoparticles. In certain embodiments, the personalizing substance isselected from the group consisting of DNA, sand, soil, metal, crematedash, ceramics, and plant tissue. In certain embodiments, thepersonalizing substance is DNA and wherein the DNA further comprises apersonal identification characteristic selected from the groupconsisting of short tandem repeats (STRs), single nucleotidepolymorphisms (SNPs), epigenetic markers, and methylated DNA patterns.In a particular embodiment, the personalizing substance is cremated ash.

In certain embodiments of the compositions described herein, the polymeris selected from the group consisting of polyvinyl acetate,polyacrylate, polymethacrylate, and copolymers and blends thereof. Incertain embodiments the polymer has a glass transition temperature thatis greater than or equal to 60° C. or has a melting point greater thanor equal to 50° C.

In certain embodiments of the compositions described herein, thepersonalizing substance is DNA, and wherein the microparticles compriseup to 0.01% (w/w) DNA. In certain embodiments, the personalizingsubstance is not DNA, and wherein the microparticles comprise up to 10%(w/w) of the personalizing substance.

In certain embodiments, the composition further comprises a tattoo ink,wherein the ink comprises at least one pigment or dye.

In some aspects, the invention relates to a method of encapsulating apersonalizing substance for administering to the skin of an individual,comprising: (a) isolating the personalizing substance from a sourceorganism or source material; and (b) encapsulating the personalizingsubstance in a non-bioerodible, polymeric microparticle, wherein themicroparticle comprises a biocompatible, hydrophobic, non-bioerodiblepolymer, and wherein the microparticle does not release thepersonalizing substance. In certain embodiments, the method furthercomprises micronizing the personalizing substance before step (b). Incertain embodiments, the method further comprises forming thepersonalizing substance into a nanoparticle before step (b). In certainembodiment, the personalizing substance is DNA. In certain embodiments,the method further comprises amplifying the DNA prior to step (b). Insome embodiments, the DNA is in liquid form prior to step (b). In someembodiments, the DNA is human DNA.

In certain embodiments of the method described above, step (b) comprisesforming the microparticles by a process selected from the groupconsisting of solvent evaporation microencapsulation, double wallformation of microspheres by solvent evaporation, hot meltencapsulation, phase separation encapsulation, spontaneous emulsion,solvent removal microencapsulation, and coacervation. In certainembodiments, a single batch of the composition is formed comprising drymicroparticles, wherein the mass of the batch of dry microparticlesranges from approximately 1 g to 3 g.

In certain embodiment, the method described above further comprises: (c)analyzing the personalizing substance isolated from the source organismor source material; (d) analyzing the personalizing substanceencapsulated in the microparticle; and (e) comparing data obtained instep (d) to data obtained in step (c) to confirm that the personalizingsubstance isolated from the source organism or source material is thesame as the personalizing substance encapsulated in the microparticle.

In some aspects, the invention relates to a method of delivering apersonalizing substance to the skin of an individual comprisinginjecting into the individual's skin a composition comprisingnon-bioerodible microparticles, wherein the microparticles comprises abiocompatible, hydrophobic, non-bioerodible polymer and encapsulatedpersonalizing substance, and wherein the microparticle does not releasethe personalizing substance. In certain embodiments, the injection ismade at the site of an existing tattoo. In certain embodiments, themethod further comprises mixing the microparticles with a tattoo inkprior to injection. In certain embodiments, the step of injecting isrepeated multiple times at different sites on the skin to form a tattoodesign. In certain embodiments, the personalizing substance is in theform of nanoparticles. In certain embodiments, the personalizingsubstance is selected from the group consisting of DNA, sand, soil,metal, cremated ash, ceramics, and plant tissue. In certain embodiments,the microparticles have a size ranging from 1 micron to 5 microns.

In certain embodiments of the method described above, the personalizingsubstance is DNA and wherein the DNA further comprises a personalidentification characteristic selected from the group consisting ofshort tandem repeats (STRs), single nucleotide polymorphisms (SNPs),epigenetic markers, and methylated DNA patterns.

In certain embodiments of the method described above, the polymer has aglass transition temperature that is greater than or equal to 60° C. orhas a melting point greater than or equal to 50° C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart showing the steps of an exemplary end-to-endprocess for delivering to a customer a tattoo that incorporates DNA ofpersonal significance. The steps performed by the customer and then bythe lab generate encapsulated DNA, alone (dry powder), or in liquid form(e.g. with a carrier solution), which is added as an additive to atattoo ink.

FIG. 2 is a flow chart showing the steps of an exemplary end-to-endprocess for delivering to a customer a tattoo that incorporates one ormore compounds of personal significance. The steps performed by thecustomer and then by the lab generate compound(s) of personalsignificance, alone (dry powder), or in liquid form (e.g. with a carriersolution), which is added as an additive to a tattoo ink.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used herein, the term “personalizing substance” refers to a materialof significance to an individual. The personalizing substance may be anatural or synthetic material, where at least a portion of the materialis capable of being encapsulated in a polymeric microparticle. The termpersonalizing substance is used herein to refer to the material bothprior to and subsequent to encapsulation.

The terms “additive” and “additive composition” are used interchangeablyherein to refer to a composition that is added to an existing tattoo orto a tattoo ink, with or without a carrier, prior to or during,tattooing.

The terms “non-bioerodible” and “non-erodible” as used herein mean inertor unreactive under physiological conditions in the skin. Thenon-bioerodible polymers and the resulting polymeric microparticlesdescribed herein are able to withstand physical dissolution and/orchemical degradation processes under the physiological environment ofbiological tissues, particularly the skin, typically for at least 5years, at least 10 years, at least 15 years, at least 20 years, or evenlonger following application to an individual's skin, such as viainjection into the skin.

As used herein, the term “hydrophobic polymer” refers to polymers thathave a low affinity for water (at physiological temperature, e.g. 37°C.) and have a lower solubility in water than polylactic acid (PLA).

As used herein, the term “high molecular weight” means a molecularweight above 10,000 Daltons (Da), preferably above 20,000 Da.

As used herein, the terms “carrier” or “additive carrier” mean acomposition for dissolving or storing a personalizing substance inencapsulated form. The carrier is typically suitable for injection intothe human skin.

As used herein, the term “encapsulated material,” means the molecularcomponents of the personalizing substance. For example, if thepersonalizing substance is sand, then the encapsulated material includessilica (SiO₂), calcium silicate (Ca₂SiO₄), calcium nitride (CaN₂),and/or silicon nitride (Si₃N₄), etc.

As used herein, the term “biological material” means any biologicalsubstance, including, but not limited to biological micromolecules, suchas a nucleotides, amino acids, cofactors, or hormones, biologicalmacromolecules, such as nucleic acids, polypeptides, proteins (forexample enzymes, receptors, secretory proteins, structural and signalingproteins, hormones, ligands, etc.), polysaccharides, and/or anycombination thereof.

As used herein, the term “physiologically compatible component” meansany component in a composition that is compatible with the physiology ofthe recipient, typically a human.

As used herein, the term “recipient” refers to the recipient of theencapsulated personalizing substance. The recipient may be any subject,human, animal or plant, capable of receiving the encapsulatedpersonalizing substance.

As used herein, “nanoparticle” refers to a particle or a structure inthe nanometer (nm) range, typically from about 1 to about 1000 nm indiameter.

As used herein, a “microparticle” is a particle of a relatively smallsize, but not necessarily in the micron size range; the term is used inreference to particles of sizes that can be, for example 1 to about 1000microns. The term “microparticle” encompasses microspheres,microcapsules and microparticles, unless specified otherwise. Amicroparticle may be of composite construction and is not necessarily apure substance; it may be spherical or any other shape.

As used herein, the term “percent loading” refers to a ratio of theweight of a personalizing substance to the weight of a microparticle,multiplied by 100.

As used herein, the term “small batch” refers to a batch size of anencapsulated personalizing substance suitable for use by no more thanone, no more than two, no more than three, no more than four, no morethan five, no more than six, no more than seven, no more than eight, nomore than nine, or no more than ten individuals, optionally with a smallamount remaining after application to the individual for verificationpurposes. In some embodiments, the batch size of the encapsulatedpersonalizing substance is less than about 10,000, 5000, 4000, 3000,2000, 1000, 500, 100, 50, 10, 1, 0.1 or 0.01 mg. Any of these values maybe used to define a range for the batch size of the encapsulatedpersonalizing substance. For example the batch size of the encapsulatedpersonalizing substance may range from about 10,000 mg to about 0.01 mg,from about 10,000 mg to about 1000 mg, or from about 5000 mg to about500 mg.

II. Compositions

Compositions for placing a personalizing substance in the skin of anindividual, to remain at the site of placement, are described herein.The personalizing substance is encapsulated in the microparticles and isalso referred to herein as “the encapsulated material”. The compositionsmay be used to personalize tattoos and/or integrate substances ofspecial significance to an individual into his/her skin.

Following delivery to an individual's skin, the encapsulated materialremains in the microparticles, and the microparticles do not erode. Theencapsulated material is not released from the microparticles. A simplein vitro test can be used to confirm that the microparticles will notrelease the encapsulated material following delivery to an individual'sskin. For example, after formation of microparticles containing anencapsulated personalizing substance, the microparticles can be immersedin aqueous solution or buffer at pH 7.4 and temperature of 37° C. in abath for at least about 1 month. Samples are removed periodically, suchas after 1 hour, after 1 day, after 1 week, and after 1 month andanalyzed using a suitable detection method to determine if any traces ofthe encapsulated material are in the aqueous solution, buffer, orsupernatant.

Suitable detection methods are known in the art. For example, suitablemethods for detection of whether any DNA is released include detectionof the fluorescence of labeled DNA released into the aqueous solution,buffer, or supernatant and/or PCR amplification of the aqueous solution,buffer, or supernatant. PCR methods for detecting low levels of DNA in asample are known in the art. See, for example, Sambrook, et al.,Molecular Cloning. (4th ed.). Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory. Conventional PCR and real-time PCR with real-timemonitoring of amplification may be used to detect any DNA release. ThePCR amplification may use the same primers and amplification conditionsas those used for amplification of DNA prior to encapsulation. The PCRamplification may follow up to 50 amplification cycles and generates adetectable number of amplified DNA molecules, if any DNA is present inthe aqueous solution, buffer, or supernatant, referred to as “theamplified product”). Following PCR, the amplified product, if present,may be detected by conventional gel electrophoresis techniques or UV-Visspectrometry for detecting double-stranded DNA. See, for example,Sambrook, et al., Molecular Cloning. (4th ed.). Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory. Presence of an amplified productfollowing the process described above indicates release of DNA from themicroparticles, and absence of amplified product indicates that DNA wasnot released from the microparticles

For non-DNA personalizing substances, suitable detection methodsinclude, IR, mass spectrometry, for example, isotope-ratio massspectrometry (IRMS) or liquid chromatography mass spectrometry (LC-MS).

As used herein the term “a microparticle that does not release thepersonalizing substance” refers to a microparticle that does not releasea substantial amount of the personalizing substance in the aqueoussolution or buffer as detected by an in vitro assay as described above.In some embodiments, the microparticle does not release a detectableamount of the personalizing substance after 1 hour, 1 day, 1 week, 2weeks, 3 weeks, 1 month, 6 months, 1 year, 5 years, 10 years, 20 years,or 30 years as determined by an in vitro assay as described above. Insome embodiments, the microparticle releases less than 10%, 5%, 1%,0.5%, 0.1%, 0.05%, 0.01%, 0.005% or 0.001% of the total amount of thepersonalizing substance contained in the microparticle. In someembodiments, the microparticle releases less than 10%, 5%, 1%, 0.5%,0.1%, 0.05%, 0.01%, 0.005% or 0.001% of the total amount of thepersonalizing substance contained in the microparticle after 1 hour, 1day, 1 week, 2 weeks, 3 weeks, 1 month, 6 months, 1 year, 5 years, 10years, 20 years, or 30 years. In a particular embodiment, themicroparticle releases less than 0.1% of the total amount of thepersonalizing substance (e.g. DNA) contained in the microparticle after2 weeks.

For compositions with DNA as the encapsulated personalizing substance,the detection method following an in vitro assay as described aboveincludes amplification by PCR followed by detection by conventional gelelectrophoresis techniques or UV-Vis spectrometry. For compositions withnon-DNA material(s) as the encapsulated personalizing substance, massspectrometry may be used as the detection method following an in vitroassay as described above.

1. Personalizing Substance

Generally, the compositions described herein include a personalizingsubstance. Suitable personalizing substances include, but are notlimited to, biological materials such as, for example, animal or planttissue, sand, soil, metal, sea water, holy water, synthetic or naturalpolymers, cremated ash, ceramics, and other physiologically compatiblecomponents. In the case of liquid personalizing substances such as seawater and holy water, lyophilization of microparticles comprising thepersonalizing substance would remove any liquid contained in themicroparticle. However, any salts or other non-volatile compoundscontained in the liquid would remain.

In some embodiments, the compositions may contain encapsulated DNAwithout any additional personalizing substances. In other embodiments,the compositions contain a personalizing substance comprising DNA andone or more additional personalizing substances comprising othercompounds. For example, the additional personalizing substances may beone or more samples from sand, soil, metal, ceramics, and/or plantproducts.

A. Exemplary Personalizing Substances

Suitable personalizing substances include, but are not limited to, sand,soil or rock particles, or compounds extracted from sand, soil or rock.

Sand consists predominately of silica (SiO₂) and other organic andinorganic minerals, such as calcium silicate (Ca₂SiO₄), calcium nitride(Ca₃N₂), silicon nitride (Si₃N₄), aluminum nitride (AlN₃), alumina(Al₂O₃), borazone “boron nitride” (BN), magnesium oxide (MgO), siliconoxysulfide (SiOS), lithium silicate (Li₂SiO₄), as well as other metaloxides/nitrides, as shown in Table 1.

The identity of personalizing substances that do not contain DNA, suchas sand, soil, metal, water, sea water, holy water, synthetic or naturalpolymers, cremated ash, ceramics, and compounds derived from plants, maybe confirmed by a suitable method, such as mass spectrometry, forexample, isotope-ratio mass spectrometry (IRMS) or liquid chromatographymass spectrometry (LC-MS).

TABLE 1 Exemplary Personalizing Substances Source for PersonalizingSubstance Personalizing Substance White Beach Quartz (SiO₂) particles ofdifferent diameter ranges and Sand limestone from coral or shells. DarkSand Quartz (SiO₂) particles of different diameter ranges and magnetite.Green Sand Quartz (SiO₂) particles of different diameter ranges andchlorite Rock Quartz (SiO₂) particles of different diameter ranges andother trace elements that vary with geographical location.

For example, the personalizing substance may contain silicon dioxideparticles extracted from a soil or rock sample. Suitable extractiontechniques are known. Following extraction, the particles may be groundby conventional means to reduce their size to less than 1 micron,optionally the particles are then screened to obtain a population ofparticles having a size range for encapsulation, or micronized toproduce nanoparticles of suitable size, typically from about 1 to about1000 nm in diameter. Optionally, the particles may be mixed withpowdered or pre-dispersed tattoo ink after encapsulation.

In some embodiments, the personalizing substance comprises particles ofa metal or ceramic object having meaning to a person receiving thesubstance. For example, such metal or ceramic objects can be ground,screened and extracted to remove unwanted components, encapsulated, andmixed with tattoo ink for inclusion in a tattoo.

In some embodiments, the personalizing substance includes extracts ofwooden items that have personal meaning to the individual. For example,in some embodiments cellulose is extracted from the wood item andencapsulated for delivery to the individual.

The personalizing substance may be added as a solid or in the form of aliquid, such as in the form of an emulsion, to the microparticle formingmaterial. Following encapsulation, the personalizing substance is in theform of small particles, typically nanoparticles, in the microparticle.Generally, the personalizing substance is in the core of themicroparticles and is surrounded by the hydrophobic, non-erodiblepolymeric matrix, i.e. the shell. The encapsulated personalizingsubstance has a size smaller than the resulting microparticles, and istypically smaller than 1 micron in diameter (or in its largest dimensionfor non-spherical particles).

B. Types of DNA Molecules in Personalizing Substances

The personalizing substances are intended to remain inert and unreactiveand to remain encapsulated following delivery to the skin.

Accordingly, in some embodiments, the personalizing substance does notcomprise a vector. As used herein the term “vector” refers to a DNAmolecule used in biotechnology for storage, propagation, delivery orintegration of recombinant DNA. Examples of vectors include plasmidbackbones, viral vectors, bacmids, cosmids, and artificial chromosomes.

Generally, the vector itself is a DNA sequence that consists of aninsert (transgene, or recombinant DNA) and a larger sequence that servesas the “backbone” of the vector. The purpose of a vector is to transferthe insert to another cell, where it may be isolated, multiplied, orexpressed. In some embodiments, the personalizing substance does notcomprise DNA that is used to transfer a DNA sequence into a cell. Insome embodiments, the personalizing substance does not comprise DNA usedfor the purpose of multiplying or expressing the genetic informationcontained within it.

C. Optional Components

1. Personal Identification Characteristics

Optionally, the compositions include one or more personal identificationcharacteristics. The one or more personal identification characteristicscontain unique information which can be used to verify that thepersonalizing substance was obtained from a particular source, e.g.,human, non-human animal, or plant. A verification step may be made priorto or subsequent to encapsulation, optionally verification may occurafter the personalizing substance is placed in the skin of anindividual.

Exemplary personal identification characteristics for DNA include, butare not limited to, microsatellite markers such as short tandem repeats(STRs) and Simple Sequence Repeat (SSR) markers, single nucleotidepolymorphisms (SNPs), and epigenetic markers, such as methylated DNApatterns. Any DNA sequence that is unique to the source organism may beused as a personal identification characteristic. For example the DNAsequence unique to the source organism may be identified by sequencingthe entire sequence of the DNA isolated from the source organism, or aportion thereof, using sequencing methods known in the art such asSanger sequencing or next generation sequencing, e.g. Illuminasequencing. DNA sequencing methods are well known in the art and aredescribed, for example, in Sambrook, et al., Molecular Cloning, (4thed.). Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory.

a. Polymorphic Genetic Markers

DNA generally includes one or more polymorphic genetic markers.Polymorphic genetic markers are highly variable regions of the genomewhich have contributed to the development of a variety of applicationssuch as forensic DNA analysis and paternity testing that are used tounambiguously identify individuals.

The identification of many polymorphic genetic markers has occurred overthe last thirty years. For example, polymorphic genetic markers known asvariable number of tandem repeats (VNTRs) are abundant and highlypolymorphic regions of DNA containing nearly identical sequences, 14 to80 bases in length, repeated in tandem. See Jeffreys et al., 1985,Nature 314: 67-73; Wyman et al., 1980, PNAS 77: 6754-6758; and Nakamuraet al., 1987, Science 235: 1616-1622. The variation in these markersbetween individuals makes them useful for identifying particularindividuals. VNTRs may be detected from small amounts of DNA usingpolymerase chain reaction (PCR). See Kasai et al., 1990, Journal ofForensic Sciences 35(5): 1196-1200. Size differences in the amplifiedPCR products are detected on agarose or polyacrylamide gels. However,the finite number of VNTRs limits the widespread applicability of thismethod, which in turn led to the identification of short tandem repeats(STR).

b. Short Tandem Repeats (STR)

STRs can be amplified by a polymerase chain reaction, and are highlyabundant and polymorphic (variable from individual to individual). STRscan contain tandem repeat sequences that differ by two (dinucleotide),three (trinucleotide), four (tetranucleotide) or five (pentanucleotide)base pairs. It is estimated that there are approximately 50,000 to100,000 dinucleotide repeats in the human genome. Trinucleotide andtetranucleotide repeats are less common; the human genome is estimatedto contain 10,000 of each type of repeat. See Tautz et al, 1989, Nuc.Acids Res. 17: 6464-6471; and Hamada et al., 1982, PNAS 79: 6465-6469.The use of tetranucleotide and pentanucleotide STRs allows betterdiscrimination of differences between individual subjects relative tothe shorter sequences. See Weber et al., 1989, Am J Hum Genet 44:388-396.

The personalizing substance may contain a human DNA sequence selectedfrom the group consisting of a dinucleotide STR, a trinucleotide STR, atetranucleotide STR and a pentanucleotide STR.

Because the size of PCR products from human tetranucleotide repeatregions typically varies between individuals, tetranucleotide repeatsare a preferred personal identification molecule for use as apersonalizing substance. For example, PCR products of two differentsizes are observed based on the inheritance for each individual of onecopy of the polymorphic marker from each parent. Each inherited copycontains a variable number of tetranucleotide repeats. Thus, twounrelated individuals likely will produce different sized PCR productsfrom the same tetranucleotide polymorphic marker. As a greater number ofdifferent tetranucleotide repeat regions are compared betweenindividuals, the probability of those individuals sharing the identicalpattern of PCR products decreases.

c. Single Nucleotide Polymorphisms (SNPs)

Single nucleotide polymorphism is a DNA sequence variation occurringcommonly within a population (e.g. 1%) in which a single nucleotide—A,T, C or G—in the genome (or other shared sequence) differs betweenmembers of a biological species or paired chromosomes. For example, twosequenced DNA fragments from different individuals, AAGCCTA to AAGCTTA,contain a difference in a single nucleotide.

SNPs may fall within coding sequences of genes, non-coding regions ofgenes, or in the intergenic regions (regions between genes). SNPs withina coding sequence do not necessarily change the amino acid sequence ofthe protein that is produced, due to degeneracy of the genetic code.

SNPs in the coding region are of two types, synonymous and nonsynonymousSNPs. Synonymous SNPs do not affect the protein sequence whilenonsynonymous SNPs change the amino acid sequence of protein. Thenonsynonymous SNPs are of two types: missense and nonsense.

SNPs that are not in protein-coding regions may still affect genesplicing, transcription factor binding, messenger RNA degradation, orthe sequence of non-coding RNA. Gene expression affected by this type ofSNP is referred to as an eSNP (expression SNP) and may be upstream ordownstream from the gene.

SNPs without an observable impact on the phenotype (so called silentmutations) are still useful as genetic markers in genome-wideassociation studies, because of their quantity and the stableinheritance over generations.

2. Nanoparticles

Optionally, the personalizing substance(s) are formed into orencapsulated in nanoparticles prior to encapsulation in the polymericmicroparticles.

Any of the aforementioned personalizing substances may be micronized toproduce nanoparticles of suitable size.

In some embodiments the nanoparticle comprises or consists of DNA from ahuman or from a companion animal. The DNA may be precipitated by calciumphosphate. In other embodiments, the nanoparticle comprises, consistsof, or is derived from non-DNA personalizing substance, such as sand,soil, metal, water, sea water, holy water, synthetic or biologicalpolymers, cremated ash, or ceramics. In certain embodiments thenanoparticles are formed by micronizing the personalizing substance toreduce its size, in preparation for microencapsulation.

The diameter of the nanoparticle may be, for example, about 1000, 900,800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30 or 20nanometers (nm). In certain embodiments, the diameter of thenanoparticle is less than about 1000, 900, 800, 700, 600, 500, 400, 300,200, 100, 90, 80, 70, 60, 50, 40, or 30 nanometers (nm). Any of thesevalues may be used to define a range for the diameter of thenanoparticle. For example, the diameter of the nanoparticle may be fromabout 20 nm to about 1000 nm or from about 20 nm to about 100 nm.

2. Polymeric Microparticles

The personalizing substance is encapsulated in a polymericmicroparticle. The core of the microparticles contains the personalizingsubstance, which is surrounded by a polymeric matrix that forms theouter shell of the microparticles.

Optionally, the personalizing substance is formed into nanoparticles,which are encapsulated in the polymeric microparticle. In someembodiments, the personalizing substance is a DNA nanoparticle which isprepared by calcium phosphate precipitation. The calcium phosphateprecipitated DNA nanoparticle may be encapsulated in a polymericmicroparticle without dissolving the DNA in a solvent.

In some embodiments, the microparticle comprises both a personalizingsubstance and a pigment or dye. Pigment or dye particles in thepolymeric microparticles are generally smaller than 100 nm andpreferably smaller than 20 nm. In some embodiments, the microparticlecomprising the personalizing substance does not include a pigment ordye.

A. Polymers

Any polymer that is hydrophobic, biocompatible, and non-bioerodible maybe used to form the microparticles. Preferably the composition andmolecular weight of the polymers that form the microparticles are suchthat the glass transition temperature of the polymers is greater than orequal to 60° C. or the melting point of the polymers is greater than orequal to 50° C. In certain embodiments, the glass transition temperatureof the polymers is greater than or equal to about 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 75, or 80° C. In certain embodiments, themelting point of the polymers is greater than or equal to about 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 65 or 70° C. Preferred polymers witha high glass transition temperature, i.e. a glass transition temperaturethat is greater than or equal to 60° C., or high melting point, i.e. amelting point that is greater than or equal to 50° C., include, but arenot limited to, poly(methyl methacrylate) (PMMA), polystyrene,polyethylene terephthalate, and polycarbonate. In a particularembodiment, the polymer is selected from the group consisting ofpolyvinyl acetate, polyacrylates, polymethacrylates, and copolymers andblends thereof. In another particular embodiment, the polymer isselected from the group consisting of polyacrylates, polymethacrylates,and copolymers and blends thereof. Preferably, if the microparticle isformed from a copolymer or blend of polymers, the copolymer or blend isformed from polymers with a high glass transition temperature or highmelting point, and does not contain any polymer with a low glasstransition temperature, i.e. a glass transition temperature lower than60° C., or a melting point that is lower than 50° C.

Suitable polymers with a glass transition temperature greater than orequal to 60° C. or suitable polymers with a melting point greater thanor equal to 50° C. include, but are not limited to, polyacrylates,polymethacrylates, polycarbonates, polypropylenes, polyalkylenes,polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates,polyvinyl ethers, polyvinyl halides, polysiloxanes, polyurethanes andcopolymers thereof, hydroxyalkyl celluloses, cellulose ethers, nitrocelluloses, methyl cellulose, ethyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose triacetate,cellulose sulphate sodium salt, poly(methyl methacrylate),poly(ethylmethacrylate), poly(butylmethacrylate),poly(isobutylmethacrylate), poly(hexylmethacrylate),poly(isodecylmethacrylate), poly(lauryl methacrylate),poly(phenylmethacrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutylacrylate), poly(octadecyl acrylate),polyethylene, poly(ethylene terephthalatepoly(vinyl acetate), and polyvinyl chloride polystyrene, and mixtures, copolymers, and blendsthereof.

Preferred polymers include polyacrylates and polymethacrylates.

In certain embodiments, the polymethacrylate is poly(methylmethacrylate) (PMMA). Medical grade PMMA (MW=35 kDa; residual MMAmonomer<0.1%) is commercially available from Vista Optics Ltd. (Widnes,UK).

B. Shapes and Sizes

The microparticles can have any shape. Typically the microparticles arespherical. Other suitable shapes include, but are not limited to,flakes, triangles, ovals, rods, polygons, needles, tubes, cubes andcuboid structures.

In certain embodiments, the microparticles have a diameter of less than10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or0.1 micron(s). Any of these values may be used to define a range for thediameter of the microparticle. For example the diameter of themicroparticle may be from about 0.1 to about 10 microns, from about 0.1to about 1 micron, or from about 0.1 to about 2 microns. Typically, themicroparticle diameter is less than 5 microns. Preferably forcompositions that are used as additives in a tattoo ink, themicroparticle diameter ranges from about 1 to about 10 microns, morepreferably from about 1 to 2 microns. Microparticles with a diameter of10 microns and less may be introduced into the skin via a tattoo gun orany similar device.

In other embodiments, larger microparticles or particles may be used.For example the microparticles may have a diameter of ranging from 10microns to 1000 microns. In these embodiments, the microparticles may bedelivered to the skin via an intradermal injection.

C. Loading of Encapsulated Personalizing Substance in Microparticles

Typically, the concentration of a personalizing substance encapsulatedin a microparticle is presented as percent loading. Because values forthe percent loading are dependent on the weights of the personalizingsubstances, percent loading values for the different personalizingsubstances may vary significantly. Therefore, different ranges for thepercent loading for different personalizing substances are contemplated.

In some embodiments, low concentrations (e.g., up to 0.1% w/w or lower)of the personalizing substance in the microparticles are required toprevent leaching of the personalizing substance from the microparticle.

In some embodiments, such as when the encapsulating material is DNA,only a small sample is provided for encapsulation. In these embodiments,the microparticles typically contain low concentrations of DNA. However,if a large amount of the encapsulating material is provided, the loadingof the encapsulating material in the microparticle can be higher as longas the resulting microparticles do not allow DNA to be released.

In some embodiments, the microparticle comprises about 0.00001, 0.00005,0.0001, 0.0005, 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% weight of theencapsulating material/weight of the microparticle (w/w). In someembodiments, the microparticles comprise less than about 0.00001,0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05,0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%weight of the encapsulating material/weight of the microparticle (w/w).Any of these values may be used to define a range for the concentrationof the encapsulating material in the microparticle. For example, themicroparticles may contain encapsulating material in an amount rangingfrom about 0.00001 to about 10% w/w or from about 0.001 to about 2% w/w.In some embodiments, the amount of encapsulating material in themicroparticles is less than about 0.1% w/w.

1. Percent Loading of DNA

Typically, percent loading for DNA in the microparticles ranges from0.000001% to 0.1% weight of DNA to the total weight of themicroparticles (% w/w). In preferred embodiments, the amount of DNA inthe microparticles is less than 0.01% (w/w) DNA, more preferably theamount of DNA in the microparticles ranges from 0.001% to 0.00001%(w/w). These loading ranges are generally applicable to single-walledmicroparticles.

However, for embodiments, in which the microparticles are double walledmicroparticles, higher loadings of DNA may be used. It is expected thatthe structure of the double-walled microparticles protects the DNA fromleaching out of the microparticles. In these embodiments, the amount ofDNA in the microparticles may range from 0.000001% to about 5% weight ofDNA to the total weight of the microparticles (% w/w), optionally fromabout 1%-5% (w/w).

2. Percent Loading of Other Personalizing Substances

Typically, the percent loading of personalizing substances other thanDNA is higher than the loadings of DNA in the microparticles. Forexample, the amount of the personalizing substance in the microparticlemay range from about 0.001 to about 10% w/w or from about 0.001 to about2% w/w. Optionally, the amount of the personalizing substance in themicroparticle is less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04,0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%w/w. Any of these values may be used to define a range for theconcentration of the substance in the microparticle. For example, theamount of the personalizing substance in the microparticle may rangefrom about 0.001 to about 10% w/w or from about 0.001 to about 2% w/w.In a particular embodiment, the microparticle comprises less than about0.1% w/w of the personalizing substance other than DNA.

3. Carrier

In certain embodiments, the compositions described herein are formulatedfor injection into the skin of a human. For example, the composition maycomprise a suitable biocompatible carrier for delivery to a human viainjection. Suitable carriers include any alcohol, including but notlimited to ethyl alcohol, isopropyl alcohol, or water. Suitable carriersalso include any combination of alcohol and water. Typically the amountof alcohol in the carrier ranges from about 5% to about 30% (w/w), andthe amount of water in the carrier ranges from about 40% to about 70%(w/w).

In preferred embodiments, the carrier is a solution of 60% water, 30%glycerin (glycerol), and 10% ethanol. Other carrier solutions including55% water, 30% glycerin and 15% ethanol; 50% water, 30% glycerin, and20% ethanol; 45% water, 30% glycerin, and 25% ethanol; or 40% water, 30%glycerin and 30% ethanol, are also contemplated.

4. Exemplary Composition Containing DNA

In certain embodiments, the personalizing substance to be delivered tothe individual, contains DNA from a human, a non-human animal (e.g. apet), or a plant.

In a particular embodiment, the DNA is from a human. No two people havethe exact same sequence of DNA in their cells. The differences in theDNA in individual humans gives rise to the unique DNA profiles that canbe used to distinguish individuals. In addition, the unique DNA profileof each individual provides a means for verifying that the personalizingsubstance is from a particular individual. Accordingly, incorporation ofDNA into a carrier or into a tattoo ink provides a unique characteristicto the tattoo ink or carrier that may be verified, for example, throughDNA sequencing or analysis of genetic markers.

The DNA may be coding or non-coding genomic DNA, coding or non-codingmitochondrial DNA or complementary DNA (cDNA). cDNA is synthesized fromRNA using reverse transcriptase. The genomic DNA, mitochondrial DNA, andRNA for synthesis of cDNA may be isolated from any organism, includingbut not limited to humans, animals, and plants. In some embodiments, theDNA is isolated from a single organism, for example, a human. In otherembodiments, the DNA is isolated from two or more organisms, forexample, two or more humans. Methods of isolating genomic DNA,mitochondrial DNA and RNA, and methods of cDNA synthesis are well knownin the art and are described, for example, in Sambrook, et al.,Molecular Cloning. (4th ed.). Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory.

D. DNA Isolation and Amplification

In some embodiments, the DNA contained in the personalizing substance isisolated directly from an organism, such as genomic DNA or mitochondrialDNA. In other embodiments, the DNA contained in the personalizingsubstance is amplified from a sample collected from the organism, forexample by polymerase chain reaction (PCR). Multiple DNA segments fortetranucleotide PCR amplification typically may be amplified in a singletube. Such multiple amplification of several DNA regions is known in theart as multiplex PCR. The multiple PCR products are separated as knownin the art, for example, by electrophoresis, and an instrument reads theelectrophoresis gel or image to automatically analyze the sizes of thePCR products. In some embodiments, the DNA contained in thepersonalizing substance is cDNA reverse transcribed from RNA isolatedfrom the organism, as mentioned above.

The DNA may be sequenced so that verification steps described below maybe performed. (Sambrook, et al., Molecular Cloning. (4th ed.). ColdSpring Harbor, N.Y.: Cold Spring Harbor Laboratory).

Preparation of DNA samples for use as a personalizing substance mayproceed as follows, although other methods of preparing analogous DNAsamples are known to the skilled artisan. One preferred method includesthe following general steps:

A sample for preparation of the DNA contained in the personalizingsubstance is collected from a sample of cheek swab, skin, hair, saliva,or blood or other tissue from an organism as is known in the art. Acheek swab sample is preferred. Protocols for collecting and handlingthe sample are known in the art.

For example, a DNA isolation kit suitable for isolating genomic DNA frombuccal cells, may be used to isolate DNA from the cheek swab. These kitsare commercially available and usually generate 0.5-2 micrograms oftotal DNA. Desirable genomic regions containing polymorphic geneticmarkers (such as STRs and SNPs) of the isolated DNA are then amplifiedvia PCR to generate micrograms, typically from 1 to 10 micrograms, ofDNA to be used as a personalizing substance. The amplified DNA may besequenced so that verification steps described below may be performed.This amplified DNA is the personalizing substance that is encapsulatedinto microparticles.

Optionally, the encapsulation of personalizing DNA molecule may includea control DNA molecule of a known sequence that is included at the sameamount as the personalizing DNA molecule. The control DNA may be usedfor testing to determine whether any of encapsulated DNA is released,such as via the in vitro method described above.

Alternatively, or optionally, the personalizing DNA may be partially orfully labeled with fluorophores, such as Alexa Fluor) dyes (MolecularProbes, Inc.). The labeled DNA may be used to confirm that the DNA wassuccessfully encapsulated, such as with flow cytometry of theencapsulated particles. Alternatively or additionally, the labeled DNAmay be used to determine whether any of the encapsulated DNA will bereleased following delivery to an individual's skin. This test may beperformed by measuring the fluorescence of the aqueous solution, buffer,or supernatant in which empty microparticles or those encapsulatinglabeled DNA were tested for DNA release in an in vitro method, such asdescribed above.

Transmission electron microscopy (TEM) may be used to verifyencapsulation of the amplified DNA.

In some embodiments, genomic DNA, mitochondrial DNA, and/or RNA isisolated from the sample using methods known in the art, such as thosedescribed in Sambrook et al. (cited above). The concentration andintegrity of the extracted DNA or RNA may be determined, for example, toinform the decision to proceed with PCR or reverse transcription or toobtain another sample.

In some embodiments, the DNA contained in the personalizing substancemay be generated by PCR. For example, DNA comprising STRs may beamplified by PCR using primers that amplify three to fivetetranucleotide repeat segments of the genomic DNA sample, optionallyincorporating a detectable label, such as a radioactive or fluorescentlabel, as is known in the art. PCR primers for amplifying the DNA may beobtained from a commercial source or may be synthesized using methodsknown in the art. Software for design of PCR primers is well known inthe art.

Examples of preferred STRs that may be amplified by PCR are set forth inTable 2 below. The skilled artisan will appreciate that additionalsuitable tetranucleotide and pentanucleotide repeats may also beamplified. One of the preferred qualities of suitable tetranucleotideDNA repeats is high heterozygosity (variability between individuals) inthe subject population. Another preferred quality of suitabletetranucleotide DNA repeats is that they do not encode a biologicallyactive product, for example, a protein, tRNA, rRNA, miRNA, or siRNA. Afurther preferred quality of suitable tetranucleotide DNA repeats isthat they do not induce an immune response and produce no therapeuticaction in the recipient.

TABLE 2 Preferred Repeats in DNA for amplification Human AlleleDistribution Number of Marker (bp) Repeats 3S1358 98 to 146 8 to 205S818 133 to 169 7 to 16 7S820 215 to 247 6 to 14 8S1179 163 to 213 7 to19 13S317 161 to 205 5 to 16 16S539 133 to 173 5 to 15 21S11 201 to 25724 to 38 8S1106 109 to 133 7 to 13 1S518 182 to 198 13 to 17 6S1017 354to 374 10 to 15 17S1304 197 to 213 10 to 14 4S2408 336 to 360 13 to 195S1467 173 to 189 8 to 12 19S245 225 to 249 16 to 22

The resulting PCR products are typically analyzed, for example, byelectrophoresis, for the successful generation of tetranucleotiderepeats and to confirm that the sample shows relatively uniquerepresentation of a DNA sample from an individual.

E. Verification of Amplified DNA

In some embodiments, the DNA is analyzed to confirm that the DNAcontained in the personalizing substance was obtained or generated fromthe desired source organism. For example, for DNA comprising STRs, thepattern of PCR products in the DNA contained in the personalizingsubstance may be compared to a control sample obtained from the sourceorganism. The DNA contained in the personalizing substance may also beanalyzed by DNA sequencing, for example cDNA sequencing or whole genomesequencing, to confirm that the DNA contained in the personalizingsubstance is from the desired source organism.

The sequencing of the DNA may be performed using methods known in theart. These include, but are not limited to basic sequencing methods,such as Sanger's method, Maxam-Gilbert sequencing and chain terminationmethods (Franga et al., Quarterly Review of Biophysics, 35(2):169-200,2002), advanced methods and de novo sequencing, such as shotgunsequencing and bridge PCR (Braslavky et al., Proc. Natl. Acad. Sci,100(7):3960-3964, 2003), or next-generation methods. Next-generationsequencing applies to genome sequencing, genome resequencing,transcriptome profiling (RNA-Seq), DNA-protein interactions(ChIP-sequencing), and epigenome characterization (de Magalhaes et al.,Ageing Res Rev. 9(3)315-323, 2010; Liu et al., Journal of Biomedicineand Biotechnology, 2012:1-11, article ID 251364, 2012; and Hall, TheJournal of Experimental Biology, 209:1518-1525, 2007). Resequencing isnecessary, because the genome of a single individual of a species willnot indicate all of the genome variations among other individuals of thesame species.

Next Generation sequencing encompasses a number of methods, including,but not limited to single-molecule real-time sequencing, massivelyparallel signature sequencing, (MPSS), Polony sequencing, 454pyrosequencing, ion torrent semiconductor sequencing, DNA nanoballsequencing, heliscope single molecule sequencing, sequencing by ligation(SOLID sequencing) and single molecule real time sequencing (SMRT).These methods are detailed and compared in Liu et al., Journal ofBiomedicine and Biotechnology, 2012:1-11, article ID 251364, 2012, andHall, The Journal of Experimental Biology, 209:1518-1525, 2007.

In some embodiments, the DNA contained in the personalizing substance isanalyzed before the personalizing substance is combined with a carrieror in tattoo ink. In other embodiments, the DNA contained in thepersonalizing substance is analyzed after the personalizing substance iscombined with a carrier or tattoo ink.

The DNA may be purified to obtain pharmaceutical/biologics grade DNAsuitably free of contaminants.

II. Methods of Making the Compositions

The microparticles may be made using a variety of knownmicroencapsulation methods, such as solvent evaporation, multi-walled(or double walled) microencapsulation, coacervation, and meltprocessing.

Any of the non-bioerodible, hydrophobic polymers discussed above may beused to form the polymeric microparticles.

1. Solvents

Solvents that may be used in forming the microparticles include organicsolvents such as methylene chloride, which leave low levels of residuethat are generally accepted as safe. Suitable water-insoluble solventsinclude methylene chloride, chloroform, dicholorethane, ethyl acetateand cyclohexane. Additional solvents include, but are not limited to,alcohols such as methanol (methyl alcohol), ethanol, (ethyl alcohol),1-propanol (n-propyl alcohol), 2-propanol (isopropyl alcohol), 1-butanol(n-butyl alcohol), 2-butanol (sec-butyl alcohol), 2-methyl-1-propanol(isobutyl alcohol), 2-methyl-2-propanol (t-butyl alcohol), 1-pentanol(n-pentyl alcohol), 3-methyl-1-butanol (isopentyl alcohol),2,2-dimethyl-1-propanol (neopentyl alcohol), cyclopentanol (cyclopentylalcohol), 1-hexanol (n-hexanol), cyclohexanol (cyclohexyl alcohol),1-heptanol (n-heptyl alcohol), 1-octanol (n-octyl alcohol), 1-nonanol(n-nonyl alcohol), 1-decanol (n-decyl alcohol), 2-propen-1-ol (allylalcohol), phenylmethanol (benzyl alcohol), diphenylmethanol(diphenylcarbinol), triphenylmethanol (triphenylcarbinol), glycerin,phenol, 2-methoxyethanol, 2-ethoxyethanol, 3-ethoxy-1,2-propanediol,Di(ethylene glycol)methyl ether, 1,2-propanediol, 1,3-propanediol,1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 1,2-pentanediol,1,3-pentanediol, 1,4-pentanediol, 1,5-pentanediol, 2,3-pentanediol,2,4-pentanediol, 2,5-pentanediol, 3,4-pentanediol, 3,5-pentanediol, andcombinations thereof. A preferred alcohol is isopropanol.

Materials that may be used to formulate a coacervate system compriseanionic, cationic, amphoteric, and non-ionic surfactants. Anionicsurfactants include di-(2 ethylhexyl)sodium sulfosuccinate; non-ionicsurfactants include the fatty acids and the esters thereof; surfactantsin the amphoteric group include (1) substances classified as simple,conjugated and derived proteins such as the albumins, gelatins, andglycoproteins, and (2) substances contained within the phospholipidclassification, for example lecithin. The amine salts and the quaternaryammonium salts within the cationic group also comprise usefulsurfactants. Other surfactant compounds useful to form coacervatesinclude polysaccharides and their derivatives, the mucopolysaccharidesand the polysorbates and their derivatives. Synthetic polymers that maybe used as surfactants include compositions such as polyethylene glycoland polypropylene glycol. Further examples of suitable compounds thatmay be utilized to prepare coacervate systems include glycoproteins,glycolipids, galactose, gelatins, modified fluid gelatins andgalacturonic acid.

3. Surfactants

Hydrophobic surfactants such as fatty acids and cholesterol may be addedduring preparation of the microparticles to improve the resultingdistribution of hydrophobic personalizing substances in hydrophobicpolymeric microparticles. Examples of suitable fatty acids includebutyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid,pelargonic acid, caprylic acid, undecylic acid, lauric acid, tridecylicacid, myristic acid, pentadecylic acid, palmitic acid, heptadecylicacid, stearic acid, nonadecanoic acid, arachic acid, isocrotonic acid,undecylenic acid, oleic acid, elaidic acid, sorbic acid, linoleic acid,linolenic acid and arachidonic acid.

Hydrophilic surfactants such as TWEEN® 20 and polyvinyl alcohol (PVA)improve distribution of hydrophilic dye in the polymers. Amphiphilicsurfactants are preferred if the dye is hydrophilic and the polymer ishydrophobic.

Surfactant such as a fatty acid or a pharmacologically acceptable saltthereof is typically added in a ratio of from 0.2 to 1 part by weight ofthe fatty acid or salt thereof to 1 part by weight of the dye.

4. Micronizing and Nanoparticle Formation

Methods for micronizing the personalizing substance for production ofnanoparticles, if needed, include, for example, sonication and/orproduction of shear forces, and rotor stator mixing or milling with aconcentric shaft, at a speed between, for example, 5,000 RPM and 25,000RPM.

In some embodiments where DNA is the personalizing substance, the DNAmay be prepared by precipitation using standard techniques, such asethanol or isopropanol precipitation, or salt precipitation. In someembodiments, the DNA is micronized by precipitation with calciumphosphate, and the precipitate is not dissolved but instead incorporateddirectly as nanoparticles into the microparticle. In some embodiments,the DNA is encapsulated as an emulsion in water which is later removedafter the encapsulation process to produce a small solid particle ofDNA. The DNA may also be bound to a solid nanoparticle such as silicondioxide or gold, or crosslinked together to form aggregates.

Methods of encapsulating DNA in nanoparticles are described in the art.See, for example, US 2009/0311295, and van de Berg et al., 2010, Journalof Controlled Release 141: 234-240.

5. Distribution of Nanoparticles within Microparticles

Preferably the nanoparticle containing the personalizing substance areuniformly distributed within the polymer microparticle and at a lowloading level to avoid any leaching of the encapsulated personalizingsubstance. This is particularly desirable when the encapsulatingmaterial is or contains DNA.

The problem with most methods of manufacture of microparticles is thatwhile the nanoparticles are dispersed initially following addition topolymer solution, the nanoparticles rapidly settle towards the bottom.Then when solvent is removed, the nanoparticles are present morepreferentially in one part of the polymer than another. It is difficultto keep the nanoparticles dispersed while at the same time removing thepolymer solvent to form the microparticles. Therefore, methods have beendeveloped wherein the nanoparticles are dispersed in the polymersolution so that the solution is “stabilized” so that the nanoparticlesstay uniformly distributed within the polymer for a period of timesufficient to form the microparticles. This time may be as short as tenminutes or as long as a few hours. The amount of time that thenanoparticle will remain suspended in the polymer depends on the sizeand composition of the nanoparticle.

Stability is a function of the selection of the polymer, the solventcomposition as well as the method of dispersion and the density of theencapsulated material. For example, the concentration of the organicpolymeric solution must be adjusted to keep the nanoparticles dispersedand prevent settling of the nanoparticles during the process ofencapsulation. In a method theoretically (if not mechanistically)analogous to beating egg whites, the polymer solution is sonicated orotherwise subjected to shear forces, using an open blade mixer or rotorstator at 5000-25,000 RPM, or milled using a concentric shaft, untilstable. Alternatively or in addition, the solvent and surfactant, ifpresent, can be used to alter the surface properties of thenanoparticles so that they remain suspended in the polymer solution. Thesolvent is then removed to form the microparticles having a uniformdispersion of nanoparticles within the polymer.

6. Methods of Making Microparticles

There are several processes whereby microparticles can be made,including, for example, multi-walled microencapsulation, hot meltencapsulation, phase separation encapsulation, spontaneous emulsion,solvent evaporation microencapsulation, solvent removalmicroencapsulation, and coacervation. These methods are known in theart. Detailed descriptions of the methods are discussed in Mathiowitz etal., “Microencapsulation”, in Encyclopedia of Controlled Drug Delivery,vol. 2, pp. 495-546, 1999, John Wiley & Sons, Inc. New York, N.Y., andare concisely presented below. A preferred method is solvent evaporationmicroencapsulation (specifically high oil to aqueous phase ratio toachieve small particles with addition of surfactant such as oleic acidto improve dispersion of the personalized fragment in the polymericphase phase). For solvent evaporation, the minimum concentration is 0.1%w/v (polyvinyl alcohol to water). Another preferred method includesaddition of the nanoparticles into the polymer liquefied by melting toensure uniform distribution.

The dispersion of the nanoparticles within the polymer matrix can beenhanced by varying: (1) the solvent or combination of solvents used tosolvate the polymer; (2) the ratio of the polymer to the solvent; (3)the size of the nanoparticle to be encapsulated; and (4) the percentageof the nanoparticle relative to the polymer (i.e. nanoparticle loading).The dispersion of the nanoparticles within the polymer matrix may alsobe enhanced by using surfactants.

In certain embodiments, the personalizing substance is analyzed duringthe process of preparing the microparticles, e.g. after micronizationand/or after encapsulation, to confirm the identity of the personalizingsubstance. Generally, the microparticles are prepared in small batches.

A. Hot Melt Microencapsulation

In hot melt microencapsulation, the personalizing substance (optionallyin the form of nanoparticles) to be encapsulated is added to moltenpolymer. This mixture is suspended as molten droplets in a nonsolventfor the polymer (often oil-based) which has been heated to approximately10° C. above the melting point of the polymer. The emulsion ismaintained through vigorous stirring while the nonsolvent bath isquickly cooled below the glass transition of the polymer, causing themolten droplets to solidify and entrap the core material.

B. Phase Separation Microencapsulation

In phase separation microencapsulation the personalizing substance(optionally in the form of nanoparticles) to be encapsulated isdispersed in a polymer solution with stirring. While continuallystirring to uniformly suspend the material, a nonsolvent for the polymeris slowly added to the solution to decrease the polymer's solubility.Depending on the solubility of the polymer in the solvent andnonsolvent, the polymer either precipitates or phase separates into apolymer rich and a polymer poor phase. Under proper conditions, thepolymer in the polymer rich phase will migrate to the interface with thecontinuous phase, encapsulating the personalizing substance (optionallyin the form of nanoparticles) in a droplet with an outer polymer shell.

C. Spontaneous Emulsification

Spontaneous emulsification involves solidifying emulsified liquidpolymer droplets by changing temperature, evaporating solvent, or addingchemical cross-linking agents. The physical and chemical properties ofthe encapsulant, and the personalizing substance to be encapsulated,dictates the suitable methods of encapsulation. Factors such ashydrophobicity, molecular weight, chemical stability, and thermalstability affect encapsulation.

D. Melt-solvent Evaporation Method

In the melt-solvent evaporation method, the polymer is heated to a pointof sufficient fluidity to allow ease of manipulation (for example,stirring with a spatula). The temperature required to do this isdependent on the intrinsic properties of the polymer. For example, forcrystalline polymers, the temperature will be above the melting point ofthe polymer. After reaching the desired temperature, the personalizingsubstance (optionally in the form of nanoparticles) is added to themolten polymer and physically mixed while maintaining the temperature.The molten polymer and personalizing substance are mixed until themixture reaches the maximum level of homogeneity for that particularsystem. The mixture is allowed to cool to room temperature and harden.This technique results in dispersion of the personalizing substance inthe polymer.

High shear turbines may be used to stir the dispersion, complemented bygradual addition of the nanoparticle into the polymer solution until thedesired loading is achieved. Alternatively the density of the polymersolution may be adjusted to prevent settling of the nanoparticle duringstirring.

E. Solvent Evaporation Microencapsulation

In solvent evaporation microencapsulation, the polymer is typicallydissolved in a water immiscible organic solvent and the personalizingsubstance (optionally in the form of nanoparticles) to be encapsulatedis added to the polymer solution as a dispersion, suspension or emulsionin an organic solvent An emulsion (i.e. a second emulsion if theencapsulating material is added as an emulsion) is formed by adding thisdispersion, suspension or emulsion to a beaker and vigorously stirringthe system. Any suitable surface active agent may be used to stabilizethe emulsion. Typical surface active agents include, but are not limitedto polyethylene glycol or polyvinyl alcohol (PVA)). The organic solventis evaporated while continuing to stir. Evaporation results inprecipitation of the polymer, forming solid microcapsules containingcore encapsulated material, where the encapsulated material is in theform of an emulsion or a solid.

The solvent evaporation process can be used to entrap a liquid corematerial in a polymer or in copolymer microcapsules, however the liquidis removed by conventional methods after the polymer has encapsulatedthe substance.

The solvent evaporation process is the preferred process forencapsulating DNA.

F. Solvent Removal Microencapsulation

In solvent removal microencapsulation, the polymer is typicallydissolved in an oil miscible organic solvent and the personalizingsubstance (optionally in the form of nanoparticles) to be encapsulatedis added to the polymer solution as a suspension or solution in organicsolvent. Surface active agents can be added to improve the dispersion ofthe material to be encapsulated. An emulsion is formed by adding thissuspension or solution to vigorously stirring oil, in which the oil is anonsolvent for the polymer and the polymer/solvent solution isimmiscible in the oil. The organic solvent is removed by diffusion intothe oil phase while continuing to stir. Solvent removal results inprecipitation of the polymer, forming solid microcapsules containingcore material.

G. Coacervation

Encapsulation procedures for various substances using coacervationtechniques have been described in the art, for example, in GB-B-929 406;GB-B-929 401; U.S. Pat. Nos. 3,266,987; 4,794,000 and 4,460,563.Coacervation is a process involving separation of colloidal solutionsinto two or more immiscible liquid layers (Ref. Dowben, R. GeneralPhysiology, Harper & Row, New York, 1969, pp. 142-143.). Through theprocess of coacervation, compositions comprised of two or more phasesknown as coacervates may be produced. The ingredients that comprise thetwo phase coacervate system are present in both phases; however, thecolloid rich phase has a greater concentration of the components thanthe colloid poor phase.

In the coacervation process, the polymer or copolymer is dissolved in amiscible mixture of solvent and nonsolvent, at a nonsolventconcentration which is immediately below the concentration which wouldproduce phase separation (i.e., cloud point). The liquid core materialis added to the solution while agitating to form an emulsion anddisperse the material as droplets. Solvent and nonsolvent are vaporized,with the solvent being vaporized at a faster rate, causing the polymeror copolymer to phase separate and migrate towards the surface of thecore material droplets. This phase-separated solution is thentransferred into an agitated volume of nonsolvent, causing any remainingdissolved polymer or copolymer to precipitate and extracting anyresidual solvent from the formed membrane. The result is a microcapsulecomposed of polymer or copolymer shell with a core of liquid material.

For example, DNA may be dissolved in water and then an emulsion of thedissolved DNA is formed in an organic polymeric solution. This emulsionis then added to aqueous solution and mixed (optionally, for DNA havinglengths of less than 2 kilobases (kb) high shear may be used) until theorganic solvent evaporates, and then the entire mixture is washed andfrozen and lyophilized, resulting in a dry particle of DNA inside thepolymer.

The material can be encapsulated using an emulsifier such as Tween 80®,oleic acid, lecithin, Brij®92, Span®80, Arlacel® 83, and Span® 85.Alternatively, the material can be encapsulated without the use of anemulsifier.

H. Multi-walled Microencapsulation

Multiwall polymer microspheres may be prepared by dissolving twopolymers in a solvent. A personalizing substance to be incorporated isdispersed in the polymer solution, and the mixture is suspended in acontinuous phase. The solvent then is slowly evaporated, creatingmicrospheres with an inner core formed by one polymer and an outer layerof the second polymer. The continuous phase can be either an organicoil, a volatile organic solvent, or an aqueous solution containing athird polymer that is not soluble with the first mixture of polymers andwhich will cause phase separation of the first two polymers as themixture is stirred.

Any two or more different non-biodegradable, hydrophobic polymers whichare not soluble in each other at a particular concentration as dictatedby their phase diagrams may be used. The multilayer microcapsules haveuniformly dimensioned layers of polymer and can incorporate a range ofsubstances.

For the preparation of double walled microspheres, each polymer isdissolved in a suitable solvent for that polymer, in separatecontainers, and mixed with surfactant such as oleic acid; thepersonalizing substance, optionally in the form of nanoparticles, isadded to one of the polymeric solutions. Then the two (or more)polymeric solutions are mixed, and the mixture is then added to a largevolume aqueous phase containing a surfactant, such as PVA, to form anemulsion (aqueous solution of water and some surfactant). High shear isapplied. The oil to water phase ratio is typically 1:20 to ensure smallmicroparticle sizes in the range of 1-5 microns, or even smallermicroparticles, such as in the range of 1 to 2 microns.

Microspheres containing a polymeric core made of a first polymer and auniform coating of a second polymer, and a substance incorporated intoat least one of the polymers, can be made as described in U.S. Pat. No.4,861,627.

I. Solvent Evaporation is Advantageous for NanoparticleMicroencapsulation

Solvent evaporation microencapsulation can result in the stabilizationof the nanoparticle in a polymeric solution for a period of timesufficient for encapsulation of the nanoparticle. In certainembodiments, the nanoparticle is stabilized in the polymeric solutionfor about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 minutes. In certainembodiments, the nanoparticle is stabilized in the polymeric solutionfor at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 minutes.In certain embodiments, the nanoparticle is stabilized in the polymericsolution for less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30minutes. Any of these values may be used to define a range for theamount of time that the nanoparticle is stabilized in the polymericsolution. For example, the nanoparticle may be stabilized in thepolymeric solution for about 10 minutes to about 30 minutes.

Stabilizing a personalizing substance within the dispersed phase(typically a volatile organic solvent) can be useful for most methods ofmicroencapsulation that are dependent on a dispersed phase, includingfilm casting, solvent evaporation, solvent removal, spray drying, phaseinversion, and many others.

By stabilizing suspended nanoparticles within the dispersed phase, thenanoparticles remain homogeneously dispersed throughout the polymericsolution as well as the resulting polymer matrix that forms during theprocess of microencapsulation.

Solvent evaporation microencapsulation has several advantages. Forexample, solvent evaporation microencapsulation allows for thedetermination of the best polymer-solvent-nanoparticle mixture that willaid in the formation of a homogeneous suspension that can be used toencapsulate the nanoparticle. Solvent evaporation microencapsulationstabilizes the nanoparticles within the polymeric solution. Thisstabilization of nanoparticles is an advantage during small scaleoperation because one will be able to let suspensions of insolubleparticles sit for short periods of time, making the process more secureand avoiding mixing between clients. Solvent evaporationmicroencapsulation allows for the creation of microparticles that haveno release of the encapsulated material. Solvent evaporationmicroencapsulation avoids the problem of “burst effect”, i.e. release ofthe encapsulated material within 1 hour of administration, which occurswith other encapsulation methods by allowing very low loading of thenanoparticles or personalizing substance and creating microparticlesthat have minimal pores.

7. Small Batch Preparation

In some embodiments, the compositions are made in small batches. Thesize of the batches may be limited by the nature and the amount of thepersonalizing substance, or by the number of end users.

In preferred embodiments, the personalizing substances are encapsulatedinto polymeric microparticles for personal use by one or fewindividuals. It is preferred, therefore, to prepare small batches ofpolymeric microparticles encapsulating the personalizing substance. Insome embodiments, the prepared batch size may be as small as for singleuse by a single individual. In other embodiments, the prepared batchsize may be as small as for single use by few, such as no more than two,no more than three, no more than four, no more than five, no more thansix, no more than seven, no more than eight, no more than nine, or nomore than ten individuals. In other embodiments, the batch size may beas small as for multiple uses by the same individual.

In other embodiments, the size of a small batch preparation may beguided by the amount of the available personalizing substance. Forexample, if DNA is used as a personalizing substance, the amount of DNAobtained from one individual through a cheek swab may only be smallenough to produce a batch for single use by a single recipient. In apreferred embodiment, a single small batch yields a sufficient amount ofmicroparticles encapsulating the personalizing substance for a singleuse by one end user.

In preferred embodiments, a small batch preparation process yieldsapproximately 1-10 g of microparticles encapsulating the personalizingsubstance, in dry form, preferably about 1-2 g of microparticlesencapsulating the personalizing substance, in dry form. For example, insome embodiments, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5,2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0 or 10 grams of themicroparticle is prepared. In some embodiments, less than 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5,5.0, 6.0, 7.0, 8.0, 9.0 or 10 grams of the microparticle is prepared.Any of these values may be used to define a range of amounts in whichthe microparticle is prepared. For example, from 0.5 to 5 grams, or from2 to 5 grams of the microparticle may be prepared. In a particularembodiment, approximately 2 grams of the microparticles is prepared.

IV. Methods of Use

The compositions described herein may be used on their own, incombination with a carrier, or as additives to substances, such asadditives to tattoo ink, for delivery to the skin, typically viainjection.

1. Use as Compositions

In some embodiments, the compositions described herein are suitable foruse as compositions of personal significance. The use of the compositionmay be chosen by the end user. For example, the compositions may be usedby the end user to preserve a substance of personal significance for along period of time. The end user may store the composition, or chose topresent the composition as a gift to another individual. Alternatively,the end user may choose to use the composition as an additive to othersubstance.

In some embodiments, the compositions are administered via injection ata desired skin site of an individual. Typically, the site contains amarking or a marking is added to the site to indicate the presence ofthe encapsulated personalizing substance in that location.

2. Use as Additives

In certain embodiments, the personalizing substances may be used as anadditive to tattoo inks. The additives may be used to create a type oftattoo having a physical connection with a person or place or event.

Obtaining a personalized ink tattoo creates a physical connection with aperson, object, place or event, because the personalized tattooincorporates into the tattoo ink and, therefore, the image displayed inthe skin, a personalizing substance.

Commercially available tattoo ink pigment typically is a composition inthe form of a powder, often comprising heavy metal salts forpigmentation. The tattoo ink pigment is dissolved in a carrier,preferably alcohol or water, for injection into the skin and todistribute the pigment within the dermis. Alcohol also may impart anantimicrobial effect. Pre-dispersed inks containing the pigmentpre-mixed with the carrier also are commercially available.

In certain embodiments, the microencapsulated personalizing substance isformulated in a dry powder form suitable for mixing with a carrier bythe tattooist. In some embodiments, the microencapsulated personalizingsubstance may be mixed with a carrier and supplied as a pre-dispersedsolution. The tattoo ink used in combination with the personalizingsubstance may be of any desired color known in the art.

In some embodiments, a tattoo ink comprising a personalizing substancemay be prepared by mixing microparticles that contain a personalizingsubstance with a tattoo ink that is suspended in a liquid such as water,glycerin or witch hazel. The microparticles and tattoo ink may be mixedby shaking, stirring, vortexing, or light sonicating of themicroparticles with the tattoo ink. In some embodiments, theconcentration of the microparticles in the mixture of microparticles andtattoo ink is 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9% or 10% w/w.

Once the microparticles that contain the personalizing substance aresuspended within the tattoo ink, the tattoo ink may be administered toan individual by pouring a small amount (for example, less than 10grams) of the microsphere-decorative tattoo ink slurry into a cup orother receptacle that is of sufficient size for one to dip a tattooinstrument that contains a tattoo needle or set of needles into the cupor receptacle. A tattoo may be created by dipping the tattoo needle intothe cup or receptacle that contains the microparticle-tattoo inkmixture, and then piercing the skin with the needle to inject themicroparticle-tattoo ink mixture into the skin.

The personalizing substance must be non-immunogenic in the recipientwhen applied to the skin. In some embodiments, the personalizingsubstance is encapsulated in a nanoparticle and/or microparticle beforeit is incorporated into the tattoo ink to prevent an immune response inthe recipient.

V. Kits

Kits for obtaining a personalizing substance from an end user and kitsfor delivering the encapsulated personalizing substance to the end userare provided. A flow chart depicting the end-to-end process of obtainingthe personalizing substance, and then isolating, preparing anddelivering the encapsulated personalizing substance to the end user ispresented in FIG. 2. A similar flow chart with DNA used as an exemplarypersonalizing substance is presented in FIG. 1.

In the process illustrated in FIG. 1, a cheek swab kit is provided to acustomer. The customer uses the cheek swab to obtain a sample from thehuman or non-human animal of interest to the customer. Then the customermails or otherwise delivers the sample to a lab. The lab isolates,amplifies (if needed), and purifies (if needed) the DNA, and thenencapsulates the DNA in microparticles. Then the encapsulated DNA islyophilized into a powder. Optionally, the powder is added to a carriersuitable for injection (not shown). Then the powder or solution isdelivered to the customer. The customer then delivers the solution orpowder to a tattoo shop, which either injects it in a desired site inthe customer or adds it to tattoo ink and creates a tattoo on thecustomer.

In the process illustrated in FIG. 2, a collection kit is provided to acustomer. The customer places in the collection vessel (e.g. a vial) asample from a source of interest to the customer. Then the customermails or otherwise delivers the sample to a lab. The lab extracts,optionally purifies and/or sterilizes one or more compounds from thepersonalizing substance, and then encapsulates the personalizingsubstance in microparticles. Then the encapsulated personalizingsubstance is lyophilized into a powder. Optionally, the powder is addedto a carrier suitable for injection (not shown). Then the powder orsolution is delivered to the customer. The customer then delivers thesolution or powder to a tattoo shop, which either injects it in adesired site in the customer or adds it to tattoo ink and creates atattoo on the customer.

In some embodiments, the kits provide the equipment for obtaining asample of the personalizing substance. The equipment may be tailored tothe nature of the personalizing substance that will be provided. Forexample, if the personalizing substance is DNA, then the kit may includea foam or cotton-tipped cheek swab, a protective container for the swab,and instructions for use. If the personalizing substance is sand, thenthe kit may include a waterproof container and instructions for use.

In other embodiments, the kits provide the final product for use by theend user. In these embodiments, the kits may include a personalizedsubstance, a carrier, and instructions for use. The kits may becustomized to the preference of the end user. For example, the kitscontain the personalizing substance in a powder form and a carrier.

In other embodiments, the kits may contain the personalizing substancepre-mixed with the carrier.

The kits may be delivered to the end user. Alternatively, the kits maybe delivered to a tattoo shop where the kit components may be used bythe end user at the tattoo shop.

EXAMPLES

Materials

Medical grade PMMA (Mw=35 kDa; residual MMA monomer<0.1%) was purchasedfrom Vista Optics Ltd. (Widnes, UK), PVA (Mw=25 kDa; 88% hydrolyzed) waspurchased from Polysciences, Inc. (Warrington, Pa., USA).dichloromethane (DCM; Burdick and Jackson, Muskegon, Mich., USA), ethylacetate (EA; Mallinckrodt, Hazelwood, Mo., USA), and 1-octanol(Sigma-Aldrich, St. Louis, Mo., USA) were analytical grade solvents.Particles were made by solvent evaporation microencapsulation.

Example 1 Preparation of Blank poly(methyl methacrylate) (PMMA)Microparticles

Materials and Methods

500 mg of PMMA (about 25,000 MW) was weighed in a 20-ml glassscintillation vial; 15 ml of dichloromethane (DCM) was added to PMMA,vortexed for 30 seconds and sonicated for 5 minutes until solutionbecame clear (1). At this point, the polymer was completely dissolvedand there was no particulate matter.

250 ml of surfactant, 1.0% poly(vinyl alcohol) (PVA) (MW=25,000 Da; 88%hydrolyzed) was poured into a 1-L Virtis® flask (2).

100 ml of 0.5% PVA (MW=25,000 Da; 88% hydrolyzed) was poured into an 800ml beaker (3). The beaker was placed under impeller (approximately 0.5cm from bottom of beaker) with a speed set at 3,000 RPM.

Virtis® Cyclone was set to “55” (13,750 RPM); then 100 microliters of1-octanol was added to the 1.0% PVA solution (2) and allowed to sit for5 minutes (4). The PMMA solution (1) was added to (4) in the 1-L Virtis®flask. This mixture was mixed on the Cyclone for 15 minutes at 13,750rpm.

The content was poured from the Virtis® flask into the 800 ml beakercontaining 0.5% PVA (3) and stirred for about 24 hours to form a slurryof particles.

The slurry of particles was poured into 50 ml Eppendorf® tubes, the capswere screwed on and centrifuged for 20 minutes at 4,000 RPM (3345×g).PVA solution was aspirated off using a 50 ml pipette tip; this solutionwas kept for further evaluation. 40 ml of distilled water was added totubes; mixed and shaken well until particles were resuspended indistilled water. The caps were screwed back on and centrifuged for anadditional 20 minutes at 4,500 RPM. Distilled water was aspirated offusing a 50 ml pipette tip. 40 ml distilled water was added to the tubes,mixed and shaken well until particles resuspended in distilled water.The caps were screwed back on and centrifuged for an additional 20minutes at 4,000 RPM (3345×g). Distilled water was aspirated off using a50 ml pipette tip. The slurry of particles was combined into one or twotubes, flash frozen and lyophilized for 48-72 hours.

Variation: The steps recited above were repeated with a different massof PMMA (about 1M MW). The only difference occurred in the formation ofthe PMMA solution.

In the variation of Example 1, 500 mg of PMMA was weighed (about 1M MW)in a 50-ml Falcon tube; 30 ml of dichloromethane (DCM) was added toPMMA, vortexed (30 seconds) and sonicated (5 minutes) until the solutionbecame clear.

Results

About 80% of PMMA used in this method formed blank PMMA microparticles.

Substantially the same yield was obtained in the variation of Example 1.

Example 2 Preparation of poly(methyl methacrylate) (PMMA) MicroparticlesContaining Low Loading of DNA and a Tattoo Ink Comprising the PMMAMicroparticles

Materials and Methods

1000 mg of PMMA (25,000 MW) was weighed in a 40-ml glass scintillationvial (1). DNA amplified at a mitochondrial locus was prepared. DNA wasextracted from harvested human buccal mucosal cells by boiling for 10minutes in the presence of 10% Chelex resin. A portion of the extractedDNA was PCR amplified using the following primers specific to anoncoding region of the human mitochondrial genome (bases 15,971-16411):

(SEQ ID NO: 1) 5′-TTAACTCCACCATTAGCACC-3′ (SEQ ID NO: 2)5′-GAGGATGGTGGTCAAGGGAC-3′

The PCR product was purified using the Invitrogen PureLink Quick GelExtraction & PCR Purification Combo kit. A portion of the purified DNAwas labeled with AlexaFluor 488, ethanol precipitated to remove excesslabel, resuspended in water, and mixed with the remaining DNA to producea solution suitable for encapsulation containing 5.6 nanograms of DNAper microliter. The DNA was dissolved in water, and the concentration ofthe solution was 5 micrograms per mL (2). About 100 microliters of DNAsolution, corresponding to 0.56 microgram, was taken for microparticlepreparation.

30 ml of dichloromethane (DCM) was added to the PMMA vial (1). 10microliters of Span® 80 (sorbitan monooleate) was added to the PMMAsolution and bath sonicated for 15 minutes (3). DNA (2) was pipettedinto the PMMA solution (3) and mixed at 10,000 rpm for 1 minute to forman emulsion (4).

250 ml of surfactant, 1.0% PVA (MW≈25,000 Da; 88% hydrolyzed), waspoured into a 1-L Virtis® flask. Virtis® Cyclone was set to 10000 RPM.250 microliters of 1-octanol was added to the 1% PVA, mixed for 1 minuteand then let to set for 5 minutes (5).

The PMMA-DNA emulsion (4) was added into the 1.0% PVA solution (5) andmixed for 15 minutes at 7,000 rpm (6).

200 ml of 0.5% PVA (MW≈25,000 Da; 88% hydrolyzed) was poured into an 800ml beaker. The beaker was placed under impeller (approximately 0.5 cmfrom bottom of beaker) and the impeller speed was set at 3,000 RPM.

The contents from Virtis® flask (6) were poured into the 800 ml beakercontaining 0.5% PVA and stirred for approximately 24 hours to form aslurry of particles.

The slurry of particles was poured into 50 ml Eppendorf® tubes, the capswere screwed on and centrifuged for 20 minutes at 4,000 RPM (3345×g).The PVA solution was aspirated off using a 50 ml pipette tip. Thissolution was kept for further evaluation. 40 ml of distilled water wasadded to tubes; mixed and shaken well, until particles resuspended indistilled water. Sonication was used, as needed, to break up anyparticle aggregates stuck to the bottom of the tubes. The caps werescrewed back on and centrifuged for an additional 20 minutes at 4,500RPM. Distilled water was aspirated off using a 50 ml pipette tip. 40 mldistilled water was added to tubes, mixed and shaken well, untilparticles resuspended in distilled water. The caps were screwed back onand centrifuged for an additional 20 minutes at 4,000 RPM (3345×g). Thedistilled water was aspirated off using a 50 ml pipette tip. The slurryof particles was combined into one or two tubes, flash frozen andlyophilized for 48-72 hours.

Results

About 60% of PMMA used in this method formed PMMA microparticles withlow amounts of DNA.

The morphology of the particles was observed using scanning electronmicroscopy (SEM). In general, the particles were spherical in shape andhad a smooth surface morphology. No pores were visible, even at highmagnification (4,000×). The microspheres generally had a particlediameter of 1-2 micrometers. No fragments of polymer or DNA wereobserved in the micrographs. Observation of these microparticles under afluorescent microscope revealed that a portion of them contained DNAlabeled with AlexaFluor 488.

Tattoo ink containing a personalizing substance was prepared by mixing 1g of the PMMA microparticles containing DNA described above with 19 g ofdecorative black tattoo ink, and shaking the mixture by hand. Thedecorative black tattoo ink comprised approximately 70% (w/w) of aliquid mixture (water, glycerin, and witch hazel) and approximately 30%(w/w) D&C Black No. 2 carbon black pigment.

We claim:
 1. A method of applying an ink tattoo to the skin of anindividual comprising injecting into the individual's skin a compositioncomprising: (i) tattoo ink; and (ii) non-bioerodible microparticlescomprising DNA encapsulated in a biocompatible, hydrophobic,non-bioerodible polymer, wherein (a) the microparticles do not release adetectable amount of the DNA for at least one year after delivery to anindividual's skin; (b) the DNA remains at the site of delivery; and (c)the microparticles comprise up to 0.01% (w/w) DNA.
 2. The method ofclaim 1, wherein the injection is made at the site of an existingtattoo.
 3. The method of claim 1, further comprising mixing themicroparticles with the tattoo ink prior to injection.
 4. The method ofclaim 1, wherein the step of injecting is repeated multiple times atdifferent sites on the skin to form a tattoo design.
 5. The method ofclaim 1, wherein the DNA is in the form of nanoparticles.
 6. The methodof claim 1, wherein the microparticles have a size ranging from 1 micronto 5 microns.
 7. The method of claim 1, wherein the DNA comprises apersonal identification characteristic selected from the groupconsisting of short tandem repeats (STRs), single nucleotidepolymorphisms (SNPs), epigenetic markers, and methylated DNA.
 8. Themethod of claim 1, wherein the polymer has a glass transitiontemperature that is greater than or equal to 60° C. or has a meltingpoint greater than or equal to 50° C.
 9. The method of claim 1, whereinthe microparticles do not comprise visible pores using scanning electronmicroscopy (SEM).
 10. The method of claim 1, wherein the microparticlesdo not comprise silica.
 11. The method of claim 1, wherein the DNA isisolated from an organism.
 12. The method of claim 11, wherein theorganism is a human.
 13. The method of claim 1, wherein the compositionfurther comprises a carrier suitable for injection into the skin. 14.The method of claim 13, wherein the carrier is selected from the groupconsisting of ethyl alcohol, isopropyl alcohol, glycerol, and water. 15.The method of claim 13, wherein the carrier is a solution of 60% water,30% glycerol, and 10% ethanol.
 16. The method of claim 1, wherein thepolymer is selected from the group consisting of polyvinyl acetate,polyacrylate, polymethacrylate, and copolymers and blends thereof.